The current concern about our global dependence on fossil fuels and the consequent impact on climate change have brought biofuels to the forefront. This interest in biofuels has prompted researchers to critically evaluate alternative feedstocks for biofuel production. One important, emerging biofuel crop is Camelina sativa L. Cranz (Brassicaceae), commonly referred to as “false flax” or “gold-of-pleasure”. Renewed interest in C. sativa as a biofuel feedstock is due in part to its drought tolerance and minimal requirements for supplemental nitrogen and other agricultural inputs (Putnam, Budin et al. 1993; Zubr 1997; Gehringer, Friedt et al. 2006; Gugel and Falk 2006). Similar to other non-traditional, renewable oilseed feedstocks such as Jatropha curcas L. (“jatropha”), C. sativa grows on marginal land. Unlike jatropha, which is a tropical and subtropical shrub, C. sativa is native to Europe and is naturalized in North America, where it grows well in the northern United States and southern Canada.
In addition to its drought tolerance and broad distribution, several other aspects of C. sativa biology make it well suited for development as an oilseed crop. First, C. sativa is a member of the family Brassicaceae, and thus is a relative of both the genetic model organism Arabidopsis thaliana and the oilseed crop Brassica napus. The close relationship between C. sativa and A. thaliana (Al-Shehbaz, Beilstein et al. 2006; Beilstein, Al-Shehbaz et al. 2006; Beilstein, Al-Shehbaz et al. 2008) makes the A. thaliana genome an ideal reference point for the development of genetic and genomic tools in C. sativa. Second, the oil content of C. sativa seeds is comparable to that of B. napus, ranging from 30 to 40% (w/w) (Budin, Breene et al. 1995; Gugel and Falk 2006), suggesting that agronomic lessons from the cultivation of B. napus are applicable to C. sativa cultivation. Finally, the properties of C. sativa biodiesel are already well described (Rice, Frohlich et al. 1997; Frohlich and Rice 2005; Worgetter, Prankl et al. 2006), and both seed oil and biodiesel from C. sativa were used as fuel in engine trials with promising results (Bernardo, Howard-Hildige et al. 2003; Frohlich and Rice 2005).
The quality of a biodiesel, regardless of its source, is dependent upon the fatty acid methyl ester (FAME) composition, which affects cold flow and oxidative stability (Knothe 2005; Durrett, Benning et al. 2008). For example, saturated FAMEs have poor cold flow properties since they can form crystals at lower temperatures, while the FAMEs from polyunsaturated fatty acids remain in solution at colder temperatures, and thus have good cold flow properties (Stournas 1995; Serdari, Lois et al. 1999). In contrast, the relationship between saturation and oxidative stability is exactly opposite that of cold flow. Fatty acid saturation is positively correlated with oxidative stability; saturated fatty acids have the best oxidative stability and fatty acids with 2 or greater double bonds have increasing oxidative instability (Knothe and Dunn 2003; Knothe 2005; Durrett, Benning et al. 2008). Additionally, polyunsaturated FAMEs can result in increased NOx emissions, e.g., NO, NO2 et al (McCormick, Graboski et al. 2001), and thus affect the production of a monitored pollutant. Very long chain fatty acids (VLCFA; as used herein, refers to those fatty acids containing greater than 18 carbons) result in a biodiesel with a high distillation temperature that does not meet existing standards (American Society for Testing and Materials, ASTM), reducing marketability. Given these trade-offs, an ideal biodiesel blend is high in oleic acid (18:1; carbons:double bonds), low in polyunsaturated FAMEs, and with few long chain FAMEs. This blend is oxidatively stable, has a low cloud point, and meets biodiesel standards (ASTM; Knothe 2005; Durrett, Benning et al. 2008).
The naturally occurring oil composition of C. sativa negatively affects its biodiesel properties. Polyunsaturated fatty acids such as linoleic (18:2) and alpha-linolenic (18:3) acids account for 52.1-54.7% of C. sativa seed oil (Ní Eidhin, Burke et al. 2003; Abramovic and Abram 2005). This likely accounts for the low oxidative stability of C. sativa FAMEs (Bernardo, Howard-Hildige et al. 2003). C. sativa seeds also contain 21.4-22.4% VLCFA, of which 11-eicosenoic acid (20:1) at 14.9-16.2% are especially abundant (Zubr 2002; Ní Eidhin, Burke et al. 2003; Abramovic and Abram 2005), likely resulting in the high distillation temperature of the FAMEs. Most desirable for biodiesel is oleic acid (18:1), which accounts for 14.0-17.4% of C. sativa seed oil (Budin, Breene et al. 1995; Zubr 2002; Ní Eidhin, Burke et al. 2003; Abramovic and Abram 2005). There is therefore the potential to optimize Camelina oil for biodiesel production by decreasing both the amount of polyunsaturated fatty acids being produced from oleic acid and decreasing the production of fatty acids with chain length of 18 carbons or greater.
Genes affecting oil composition are well characterized in Arabidopsis thaliana, a close relative of Camelina sativa, as well as in some other plants. For example, oleic acid (18:1) is converted to linoleic acid (18:2) in the endoplasmic reticulum by the membrane bound delta-12-desaturase FATTY ACID DESATURASE 2 (FAD2). In Arabidopsis fad2 mutants, levels of 18:1 oleic acid in the seeds increase by a factor of 2-3.4 while levels of 18:2 linoleic acids are decreased by a factor of 4-10 (Okuley, Lightner et al. 1994). Thus, mutations affecting FAD2 have been shown to lead to higher levels of oleic acid in A. thaliana and other studies have shown FAD2 has a similar role in crops such as canola (Hu, Sullivan-Gilbert et al. 2006), sunflower (Hongtrakul, Slabaugh et al. 1998) and peanut (Patel, Jung et al. 2004).
Very long chain fatty acids are formed in the cytosol of A. thaliana by sequential addition of 2 carbon units to 18 carbon fatty acid CoA conjugates. The rate limiting step is thought to be initial condensation, catalyzed in the seed by FATTY ACID ELONGASE 1 (FAE1) (James Jr, Lim et al. 1995) (Kunst, Taylor et al. 1992). In wild type Arabidopsis, approximately 25% of fatty acids in seeds are long chain fatty acids, while fae1 mutants contain less than 1% long chain fatty acids. Interestingly, 18:1 content in seeds increases by a factor of 2 in A. thaliana fae1 (Kunst, Taylor et al. 1992). In Brassica napus, reductions in long chain fatty acids, particularly erucic acid (22:1), are linked to changes in FAE1 activity (Han, Labs et al. 2001; Katavic, Mietkiewska et al. 2002; Wang, Wang et al. 2008; Wu, Wu et al. 2008).
The close relationship between A. thaliana and C. sativa suggests that FAD2 and FAE1 may play similar roles in both species, making these genes good targets for manipulation of oil composition in C. sativa. To our knowledge, FAD2 and FAE1 gene sequences have not been previously reported for C. sativa. Indeed, published studies detailing the biology of C. sativa and its closest relatives in the genus Camelina are few. However, several important findings can be drawn from the literature. Taxonomic treatments describe 11 species in the genus with a center of diversity in Eurasia (Akeroyd J: Camelina in Flora Europaea. 2nd edn. Cambridge, UK: Cambridge University Press; 1993.) although C. sativa, C. rumelica, C. microcarpa, and C. alyssum are naturalized weeds with broad distributions. Camelina species can be annual or biennial, with some species requiring vernalization to induce flowering (Mirek Z: Genus Camelina in Poland—Taxonomy, Distribution and Habitats. Fragmenta Floristica et Geobotanica 1981, 27:445-503). Chromosome counts range from n=6 in C. rumelica (Brooks R E: Chromosome number reports LXXXVII Taxon 1985, 34:346-351; Baksay L: The chromosome numbers and cytotaxonomical relations of some European plant species. Ann Hist-Nat Mus Natl Hung 1957:169-174.) or n=7 in C. hispida (Maassoumi A: Cruciferes de la fore d'Iran: etude caryosystematique. Thesis. Strasbourg, France, 1980.), upwards to n=20 in C. sativa, C. microcarpa, and C. alyssum (Gehringer A, Friedt W, Luhs W, Snowdon R J: Genetic mapping of agronomic traits in false flax (Camelina sativa subsp. sativa). Genome 2006, 49:1555-1563; Francis A, Warwick S: The Biology of Canadian Weeds. 142. Camelina alyssum (Mill.) Thell.; C. microcarpa Andrz. ex DC.; C. sativa (L.) Crantz. Canadian Journal of Plant Science 2009, 89:791-810). Some Camelina species are interfertile; crosses of C. sativa with C. alyssum, and C. sativa with C. microcarpa, produce viable seed (Tedin O: Vererbung, Variation and Systematik in der Gattung Camelina. Hereditas 1925, 6:19-386). More recently, plastid simple sequence repeat (SSR) markers (Flannery M L, Mitchell F J, Coyne S, Kavanagh T A, Burke J I, Salamin N, Dowding P, Hodkinson T R: Plastid genome characterisation in Brassica and Brassicaceae using a new set of nine SSRs. Theor Appl Genet. 2006, 113:1221-1231.) and randomly amplified polymorphic DNA (RAPD) markers have been reported and a mapping study using amplified fragment length polymorphisms (AFLP) has been published (Gehringer A, Friedt W, Luhs W, Snowdon R J: Genetic mapping of agronomic traits in false flax (Camelina sativa subsp. sativa). Genome 2006, 49:1555-1563). Additionally, the sequences of a few C. sativa transcription factors are available from the literature (Martynov V V, Tsvetkov I L, Khavkin E E: Orthologs of arabidopsis CLAVATA 1 gene in cultivated Brassicaceae plants. Ontogenez 2004, 35:41-46.) and in GenBank.
As an oilseed crop in the Brassicaceae family, Camelina sativa has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken by the present inventors to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1.